Resolution-increasing methods in microscopy are known in which the sample is illuminated so that a region is created that can be detected by fluorescence and which is smaller than a region corresponding to the diffraction limit according to Abbe.
This is possible by a non-linear interaction according to various methods:                Excitation of previously excited molecules by stimulated emission (STED, Klar and Hell, Opt. Lett. 24 (1999) 954-956)        Excitation of previously excited molecules by further excitation to a higher, non-fluorescent state (Excited State Absorption, Watanabe et al., Optics Express 11 (2003) 3271)        Depopulation of the ground state by triplet occupation (Ground State Depletion, Hell and Kroug, Appl. Phys. B 60 (1995) 495-597)        Switching of a dye between a fluorescent state and non-fluorescent state, less fluorescent state, or fluorescent state characterized in some other way (such as, different emission wavelength, polarization) (Hell, Jakobs, and Kastrup, Appl. Phys. A 77 (2003) 859-860)        
So-called photo-activation localization microscopy (PAL-M) is based on the possibility of localizing individual molecules with significantly more precision than the full width half maximum of the point-spread function (PSF) when no other emitting molecule is located in the direct neighborhood. This could be guaranteed in optically switchable molecules such that the activation radiation (that sets the molecule into a state suitable for fluorescence excitation) has a suitably low intensity. The excitation radiation then excites only molecules with low density to fluorescence. From the individual PSF imaged on the detector, the locations of the molecules can then be determined with an accuracy limited by the number of photons and the noise. The original state is re-established through the reversible or irreversible switching back to the non-fluorescent excitable state and the process can be repeated until a sufficient, high-resolution image of the sample exists.
The so-called PAL-M method is described in WO2006/127692.
The method involves the photo-activation of individual molecules separated from each other according to the expansion of the detection PSF and their high-precision localization by fluorescence detection.
The PAL-M method as described in WO2006/127692 uses the following primary steps to generate a microscopic image with an optical resolution increased relative to a standard microscope:                1. Photo-activation of individual molecules: Through the activation, the fluorescence properties of the molecules are changed (turning on/off, changing the emission spectrum, . . . ), wherein the activation is performed so that the distance between activated molecules is greater than or equal to the optical resolution of the standard microscope (given by Abbe's resolution limit).        2. Excitation of the activated molecules and localization of the molecules with a spatially-resolving detector.        3. Deactivation of the activated molecules.        4. Repetition of steps 1-3 and superimposition of the localization points from step 2 that were obtained from different iteration steps to form a high-resolution image.        
The activation is performed advantageously, but not necessarily, using far-field illumination and has a statistical distribution.
Substances are used (designated as “optical labels”) that can be transitioned from an inactive state to an activated state.
These substances, called “PTOL,” are activated by a suitable activation radiation. First, the sample is activated such that only a sub-set of the marking molecules in the sample are activated, that is, can emit fluorescence radiation.
Then the excitation radiation is applied to the sample and the fluorescent sample is mapped, wherein the fluorescence radiation can then originate only from the activated sub-set.
After this fluorescence has died down, a new activation is performed that now detects a different sub-set of the PTOL for statistical reasons.
Thus, the set of all marking molecules is divided into different sub-sets that are transitioned one after the other into the state that can be excited to fluorescence. After the excitation, the fluorescence radiation is imaged, advantageously with a sensor array.
A corresponding fluorescence microscope has an excitation light source and also a switching light source that load the sample with corresponding signals.
The sample is imaged by means of an objective lens onto a sensor array.
A computer is provided that actuates the switching light source and the excitation light source by means of control lines.
Here, the activation and excitation can take place in total reflection (FIG. 1, TIRF=total-internal-reflection fluorescence) or in the far field (FIG. 2).
In this method, the resolution is determined above all by the local photon statistics at the location of the molecule. This is related to the total number of detected fluorescence photons, the local intensity, and the background.
An optimization of these statistics through or after the detection leads directly to an improved localization accuracy and thus an improved resolution.
Alternatively, such optimized statistics could allow a quicker image recording at the same resolution.
In addition to the resolution, another important feature is the possibility of detecting several different molecules in one sample. This allows correlations between molecules and structures in the sample to be studied. In order to produce these correlations with a high locational accuracy, a simultaneous detection of the signals on a detector is a preferred solution.